UDC 581.1
APPLICATION OF CELLULAR BIOTECHNOLOGY FOR STORAGE OF
ASPEN BIODIVERSITY (Populus tremula L.)
Lyutsiya Aubakirova*, Elena Kalashnikova ***
* Eurasian national university named after Lev Nikolaevich Gumilev,
010000, Astana, Republic of Kazakhstan, web: www.enu.kz
*** Russian State Agrarian University –MSAU named after Konstantin
Timiryazev, web:www.timacad.ru
The technology of clonal micro propagation of the Populus tremula L. at
the expense of activation the meristem existing in a plant and induction of
adventives buds’ formation from primary and graft callus tissue is elaborated.
Key words: the Populus tremula L, crop of a tissue, micro sprouts, adventives
buds, callus genesis, meristem.
Aspen (Populus tremula L.) - member of the genus Populus (poplar), is a
tree specie widely spread in Russia, growing on a wide variety of soils, both in
pure plantations and in mixed forests. For the Republic of Kazakhstan Aspen is
one of the most important wood forming species, where it presents by itself the tree
of the first magnitude: its height is up to 35-40 m, diameter is up to 80-90 cm. In
the forests of the Republic of Kazakhstan aspen plantations grow in the area of 238
thousand hectares, which is more than 21% of the area covered by forest
vegetation. Despite of the fact that nowadays more than 14 thousand hectares
(more than 6% of the breed) took over-mature aspen, its annual average growth in
the forests of the republic more than 4.7 m/ha. Current growth plantations of the
best forms often exceeds 20 m 3 / ha.
Until recently, the aspen were viewed as "secondary" low-grade species,
often rotten from the first years of its life. Due to this in many instructions it is
recommended to replace aspen plantations with pine or oak plantations. Currently,
however, in most countries the major challenge is to grow the forest fast growing
trees to produce wood for pulp and paper, chemical and lumber industries.
Fast-growing species allow preserving a balance between consumption and
the reduction of wood pulp for many years. Great significance in the normalization
of the balance between consumption and reproduction of forest plantations from
becoming fast growing species of wood. At the present time in Russia and another
foreign country have developed technologies for cultivation of such plantations.
To the establishment of plantations of energy forests great attention was paid
by many European countries - Britain, France, Germany and others. In Kazakhstan
while energy plantations are still poorly developed. In the stage of experimentalindustrial are located power stations, for which the organized cultivation of energy
forests, that is, working for the burning of wood. Widely used waste products of
wood recycling and lumbering and also energy peat for the production of
properties of heat and power energy (countries of Scandinavia) (Alekseev,
Krasovskaya, 1995).
Awareness of the problem of energy security in the world first occurred in
connection with the repeated increases in oil prices.
Particularly great importance of aspen tree as of a fast-growing tree species,
easily reviving in natural way. Its stands’ age of maturity comes in 2-2,5 times
faster in comparison with the coniferous and oak species. This allows using wood
efficiently in the quality of heat sources. The cost of cultivation of aspen wood is
cheaper than pine, fir and oak, and consequently the demand for aspen wood will
grow. However, the main plantations of aspen in the Republic of Kazakhstan is
characterized by extremely low marketability, due to the defeat of fungal diseases,
causing for rot and reduces in the quality of the wood. At the same time, there are
clones of aspen which are resistant to fungal diseases, and characterized by full
arborous trunks and high productivity.
With the increase of anthropogenic impact of human activities on the
environment leads to the sharp rise of biodiversity of vegetable, as in the level of
species and of ecosystem. The burning issue of the day in the republic is the
preservation of different types of aspen (clone) and the diversity of stable aspen
phytocenosis.
One of the promising methods for the preservation biodiversity of plants is
the application of biotechnology, cell and particular method of clonal micro
propagation, significantly reduces the time needed to obtain high-quality of the
material (Kalashnikov, 2009).
Materials and Methods. As an object of research served one-year sprouts of 1520 cm long, isolated from the crone of an adult tree of the Populus tremula L.
Sprouts contained top and axillary sleeping buds. Cut buds were placed in a vessel
withwater and kept in the room temperature until the stage of a green cone’s and
young sprouts’ occurrence, which was further, used for introduction to the
culture in vitro [4].
Sprouts were processed with the use 96 % spirit, after what they were
divided into segments (1-1,2 cm) and put in the saturated solution of hypochlorite
sodium for 30-45 minutes or in 3 % solution chloramines. Further, the sterilized
segments were washed out with the use of the sterile distilled water and put it into
sterile nutrient medium of Murasiga and Skuga [5] containing BAP(three times 6benzilaminopurin) in the concentration of 0,5 mg/l and 2,4-D (2,4-dichlor
phenolacetic acid) 0,2 - 1,5 mg/l or IAA (0.5 mg/l) and NAA(naftilacetic acid) 0,2
mg/l inducing the process of callusogenesiss or the activation of developed existing
meristems. [6, 7].
Explants grew up plants in a light room, where the sustained temperature
was about 26°С and 16-hours photoperiod and 3 thousand lux intensity of light
were supported. [8].
For the regeneration of sprouts from the callus tissue there applied a
nutrient medium, containing mineral salts under the prescription of Murasiga and
Skuga or WPM with an addition of various cytokine to regeneration of
runaways (BAP, kinetin) - 2,0 mg/l in a combination with 2,4-D, NAA or IAA in
the concentration of 0,5 mg/l.
The rootages of micro sprouts in vitro were spent in a nutrient
medium WPM containing IMK in the concentration of 3 mg/l.
Results and discussion. Researches have shown that in the process
of cultivation explants in vitro within 3-4 weeks in a nutrient medium MC,
containing 2,4-D and BAP there was observed the formation unorganized growing
callus tissues fabrics of friable type in a basal parts of a sprout. It is experimentally
fixed that the process of callusogenesis is in direct dependence on the
concentration 2, 4-D in a nutrient medium. However, with the raised concentration
of the given auxin there observed the decrease of a considered indicator (Tab. 1)
which is revealed by the intensity in growth of callus tissue.
Tab. 1 The influence of various concentrations 2,4-D on callusogenesis
in the crop of Populus tremula L tissues
Concentration 2,4D, mg/l
The formation of
a callus, %
Increment of callus
tissues, mg
0,2
0,5
0
40,18,2
0
39,71,4
1,0
66,79,8
62,02,1
1,5
43,47,9
56,01,2
Thus, it has been revealed that the basic condition for the callusogenesis in
the crop of Populus tremula L tissues is an addition exogenous auxin 2, 4-D in
concentration of 1 mg/l.
As the main factor, initiating secondary differentiation of cells is the balance
of phytohormones , the influence of auxins- indolylasetic acids (IAA) has been
investigated; naftilasetic acids (NAA); 2,4-dichlorfenolasetic acids (2,4-D) and
cytokine - 6-furfuralaminopurine (kinetin) and 6-benzilaminopurine (BAP) to the
formation morphogenetic structures in the callus tissue of the Karelian birch. As an
initial material heterogeneous globular calluses of green color were used. As the
basic environment МСenvironment was used. The account of the received data
was drawn in 30 days from the moment of the cultivation of callus tissues in
inducing environments (Tab. 2).
Tab. 2 Influence of phytohormones (mg/l) to the morphogenesis of the
crop of Populus tremula L tissues.
Considered
indicators
Kinetin 2
IAA
NAA
0,5
0,5
The number of
morphogenetic
42,33,7
callus , %
Average
number
of
4,60,4
adventives
buds for 1
callus, piece
BAP 2
2,4D 0,5
IAA
NAA
0,5
0,5
2,4D 0,5
33,22,6 12,32,7 73,66,4 45,34,7 15,74,3
3,70,3
3,30,7
8,10,9
4,71,3
4,01,0
After four-week cultivation from callus tissue there started the formation of
morphogenetic structures presented by dense dark- green areas (meristematic
centre), consisting of a cone of increase and leaf rudiments. Further plantsregenerators were formed from them. From the table 2 it follows, that the
maximum increase in coefficient of reproduction is reached by addition to the
nutrient medium IAA in concentration of 0, 5 mg/l and BAP 2 mg/l. The average
of the induced buds de novo in this variant makes 8, 1 pieces on one callus. In
other variants considered indicators were for 1, 5-2 times lower, and generated
adventives buds distinguished with reduced growth.
Repeated cultivation of callus tissue on investigated variants of nutrient
mediums again led to the formation of adventives on the average of 6-8 pieces for
one callus, from which, in consequence, micro sprouts were formed.
Generated sprouts propagated with the use of the method of activation of
development in a plant meristem (cutting) which is based on removal of apical
dominance. Thus divided into the segments, containing two buds and cultivated on
non hormonal to a nutrient medium with half maintenance in its structure of macro
salts under the prescription WPM or Murasige and Skuga. In these conditions an
active growth of buds and formation of the micro sprouts characterized by normal
morphology are observed. FiG.1
A
B
FiG. 1 - Development of the aspen micro shoots of axillary buds: A - WPM, B Murasige and Skuga
The
multiplied
micro
sprouts
implanted
on
nutrient
medium WPM containing IAA in concentration of 3 mg/l. The percent of the root
plants has made 85, 3 %.
Thus micro cuttings received in this way were transferred to rooting medium
containing IBA at a concentration of 2 mg / liter. Cultivation of microshoots during
1-1,5 months in these conditions contribute to gross-perpetuating of microshoots,
which later were ready to transfer to the conditions in vivo (FiG. 2). The entire
cycle of micro propagation realized in a light room where the temperature was 23,
24-hour photoperiod and the light intensity of 1250 lux.
Formed aspen plants under in vitro conditions differ on biometric indicators,
despite the same period of their growth in vitro. Plants regenerated the triploid
forms were characterized by more rapid growth of (2,0-5,5 cm height) and 95%
had well-developed root system.
FiG. 2. - Tube plants of aspen
An adaptation vitro plant was carried out in greenhouses, which were
equipped with the installation of artificial fog. Relative humidity ranged from 90%
to 65% as the acclimation of plants. Our experience has shown, the term transplant
plants from the conditions of the in vitro conditions in vivo, has a significant
impact on the survival rate of test-tube plants. The best time to transplant test-tube
plants is spring or early summer. Plants with 2-3 leaves and well developed root
system capable of adapting to the conditions in vivo. Regenerated plants were
grown in pricked out packets or transferred directly into the soil, consisting of
accessible forms of nitrogen (NH4 + NO3) - not less than 130mg/kg, phosphorus
(P2O5) - not less than 250 mg / kg of potassium (K2O) - not less than 400 mg / kg,
the mass fraction of moisture - not more than 65%, the pH of saline suspension 6,6. Fertilizing of plants was conducted daily with mineral fertilizers "Kemira lux”
by adding a chalk or lime.
Equally important, in our view, in vitro adaptation of plants to soil
conditions has the height and physiological state (e.g., degree of rooting) of
regenerated plants. The received data testifies about the existence of genotypic
dependence and the ability of plants to adapt to the conditions in vivo. It was
established that the triploid aspen were characterized by high adaptative capacity
(65%) compared with diploid forms (30%) and had a high shoot 2 times larger than
the diploid form.
Adapted plants reached a height of 25-30 cm were carried to the area of
plantations. An inventory of uterine plantation in the end of September showed that
the growth of seedlings in height of open ground for triploid and diploid forms of
aspen was 25.8 and 15.2 cm respectively.
Thus, in vitro methods to allow receiving successfully sufficient quantity of
planting material of highly productive aspen, and resilience to the core rot.
Potential method is far from exhausted, and some of its stages can still be
improved. Genetic identity of planting material with maternal copy allows carrying
forestry practically on the basis of grain - to grow plants with known properties
(Shabunin, Podolskaya, 2005). The most promising in terms of the Republic of
Kazakhstan
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